How the volatile organic compounds emitted by corpse plant change through flowering

The corpse plant (Amorphophallus titanum) is so named because it produces a pungent, foul odor when flowering. Little is known about how the emitted volatiles change throughout the two-day flowering period. In this study, the comprehensive monitoring of the presence and change in volatile molecules during the female and the male flowering phases of A. titanum was conducted, and the plant temperature was monitored. A total of 422 volatile features were detected over the entire sampling period, of which 118 features were statistically significantly different between the pre-flowering and both flowering phases, and an additional 304 features were found present throughout the flowering period. A total of 45 molecules could be assigned putative names. The volatile profile of A. titanum changes over the two-day flowering period, with the S-containing molecules and aldehydes dominant in the female flowering phase, and the alcohols and hydrocarbons dominant in the male flowering phase. The two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS) enabled us to identify 32 new molecules produced by A. titanum. Each of these molecules alone, and in combination, likely contribute to the different odors emitted during the flowering phase of A. titanum.

When a plant is unable to fertilize itself effectively, it uses another method, such as the wind, insects, or birds to help it reproduce. A plant can attract insects and birds via the use of flower shapes, flower color pattern, and emission of odors 1 . One fascinating example of the effective use of odor attraction for the purpose of using insects to fertilize flowers is by the Amorphophallus titanum, a native of Western Sumatra and Western Java. A. titanum has the largest unbranched inflorescence in the world 2 , which makes it public favorite in botanical gardens around the world. The spadix of A. titanum extends up to 2.5 m in height and is dull yellow in color. The spathe is about 3 m in circumference, about 1.6 m in height, and is pale green in color with white spots on the outside and purplish-wine lines on the inside 3 . A. titanum is economically important for food and medicinal purposes. The underground storage stem (tuber) of A. titanum contains more glucomannan than the other Amorphophallus species, which helps in reduction of cholesterol levels, obesity and diabetes [3][4][5] .
When flowering, A. titanum emits a decay-like stench during its two-day flowering period 6 . The pulsing waves of pungent odors produced by a flowering A. titanum has led to it being referred to as the "corpse plant" 7 . The most common odors describe it as smelling like a rotting animal, a dead mouse, foul, and sulfur-like during flowering. Though produced simultaneously, the individual volatile molecules emitted during female flowering include: dimethyl disulfide (garlic-like odor 8 ), dimethyl trisulfide (foul odor 9 ), methyl thioacetate (sulfurous odor 10 ), and isovaleric acid (cheesy, sweaty odor 10,11 ). The diffusion of volatile molecules from the flowers is enhanced by thermogenesis. The spadix thermogenesis period starts after the opening of the spathe on the first day, reaching 36 °C, in pulses, synchronizing with the waves of the carrion-like odor 12 . The thermogenesis of male flowers begins on the second day when pollens are being released, where the temperature of the florets can also reach up to 36 °C 13 . The flowering A. titanum draws insects that are typically attracted to carrion, including dung beetles and flesh flies 9 .
Little is known about the variety and abundance of the volatile molecules and odors emitted by A. titanum over time and publications appear to only focus on the female flowering phase 8,10 . In the work presented here, we undertake a comprehensive survey of the molecules produced by A. titanum before and during the female and the male flowering phases, an effort complimented by thermogenesis measurements.
Processing and analysis of chromatographic data. Chromatographic data were processed and aligned using ChromaTOF. For peak finding, a signal-to-noise cutoff (S/N) was set at 100:1 (a minimum of three apexing masses) in at least one chromatogram and a minimum of 50:1 S/N in all others. The resulting peaks were identified by a forward search of the NIST 2011 library. For putative peak identification, a forward match score of ≥ 850 (of 1000) was required. For the alignment of peaks across the chromatograms, the maximum 1D and 2D retention time deviations were set at 7.5 s and 0.15 s, respectively, and the inter-chromatogram spectral match threshold was set at 600. The molecules eluting prior to 200 s in the first dimension and 0.4 s in the second dimension were removed prior to statistical analysis using the Classification feature in ChromaTOF 15 . A data cleaning step was performed to remove suspected chromatographic artifacts and common environmental contaminants, as defined previously 16 . Statistical analyses. All statistical analyses were performed using R v3.5.3 (R Foundation for Statistical Computing, Vienna, Austria). Prior to statistical analyses, the relative abundance of molecules across chromatograms was normalized using probabilistic quotient normalization (PQN) 17 and peak intensities were log-transformed, mean-centered, and then unit-scaled. The Mann-Whitney U test 18 was used to select volatile molecules that were statistically significantly different between the flowering phases, with a p-value of 0.05 as the threshold for statistical significance.
Ethics statement. We declare that the work reported here is consistent with the IUCN Policy Statement on Research Involving Species at Risk of Extinction.

Results and discussion
Observation of temperature change during flowering. The A. titanum (Fig. 1a-c) was sampled for temperature and volatile molecules over four days. Thermal profiling was used to help determine the onset and end of the female and male flowering periods 12,13 (Fig. 1d, e). The highest female and male flowering temperatures were 35.6 °C and 33.2 °C, respectively. The maximum measured temperature during male flowering was 2.8 °C lower than expected 13 , either due to our 2 h sampling window being too coarse, causing us to miss the maxima, or due to a small window cut by the greenhouse staff to conduct hand pollination. The designation of the onset and end of the female and male flowering is projected onto Fig. 1f. The volatile molecules sampling was staged into three categories: pre-flowering, female flowering, and male flowering. All other samples were considered transition and not included in statistical analyses.  were statistically significantly different between the pre-flowering and the female flowering samples (p ≤ 0.05). Putative names could be assigned to six features (Table 1), of which one feature, identified as limonene, has been detected for the first time in A. titanum. The change in abundance of these six molecules over the entire sampling period is visualized using a heat map (Fig. 2), which shows five of the six being more abundant during female flowering, with sulfur-containing molecules dominating. During the female flowering phase, dimethyl disulfide (DMDS) was present in the highest abundance. DMDS was also detected, albeit at a lower intensity and infrequently, in the pre-flowering and the male flowering phase (Fig. 2). DMDS has been previously reported as a dominant chemical component of A. titanum scent, with an odor generally described as garlic-like [8][9][10]19 . Dimethyl trisulfide (DMTS) was present in the second highest relative abundance, with peak emission during the female flowering phase and decreasing during the female to male transition. DMTS was not detected during the male flowering phase (Fig. 2). DMTS's decayed cabbage, decayed meat and garlic smell is considered a main odor contributor for A. titanum 8,10,19 with a very low odor threshold 20 .

Volatile profiles of
Methyl thiolacetate was the third most abundant molecule, followed by methanethiol and 3-methylbutanal. The level of these three molecules was highest during the female flowering phase and was lower or undetected in the other phases (Fig. 2). Methyl thiolacetate has a cheesy, garlic smell 21 and has been detected in the floral scent of A. titanum 10 . Methyl thiolacetate in combination with DMTS was attractive to adult carrion beetles (Necrophila americana and Oiceoptoma noveboracense) 22 . Methanethiol (sulfurous odor 23 ) was released less than the other S-containing molecules (Table 1). Methanethiol is the precursor for biosynthesizing DMDS in plants 24 . 3-Methylbutanal was present in high abundance during the female flowering phase, and remained somewhat abundant thereafter (Fig. 2). 3-Methylbutanal (malty odor 23 ) has been detected in the headspace of the fluid secreted from the spadix of A. titanum during female flowering 10 . Limonene was most abundant during the preflowering period, reaching its maximum just prior to the start of the female flowering phase and then dropping to barely detectable levels thereafter (Fig. 2). Although the citrus-scent of limonene has been detected in the www.nature.com/scientificreports/ floral scent of other Amorphophallus species, such as A. arnautovii, A. maximus and A. Zenkeri 8,9 , it is possible that the limonene monitored here came from a geranium flowering nearby at the time 25 . One hundred features were statistically significantly different between the female and the male flowering phase samples (p ≤ 0.05). Of these, five features were overlapping with those found statistically significantly different between the pre-flowering and the female flowering samples, and 95 features were found new in this comparison. Of the 100 features, putative names could be assigned to 22 molecules (Table 1), among which seven (S-containing and aldehydes) molecules were found dominant in the female flowering phase (Fig. 2). Among the S-containing molecules, DMDS, DMTS, methyl thiolacetate and methanethiol, which were dominant in the female flowering phase in comparison to the pre-flowering phase, remained statistically significant and dominant in the female flowering phase in comparison to the male flowering phase, with the presence of dimethyl sulfide (cabbage-like odor 23 ) being a new addition to this family. DMDS and methanethiol were present in lower abundances during the male flowering period but still likely contributing to the rotting/dead animals smell during that period as they have a low odor threshold. Aldehyde emission patterns were similar to the S-containing molecules, and had higher abundances in the female flowering phase. Nonanal (citrus and fatty odor 26 ) and benzaldehyde (almond odor 26 ) have been found previously in the appendix of A. titanum 21 . Benzaldehyde has been found attractive to Diabrotica virgifera beetle 27 .
Dominant volatile molecules exuded during the male flowering phase. Among the 22 putatively identified molecules, which were statistically significantly different between the female and the male flowering samples, 15 molecules were found dominant in the male flowering phase. Their abundance is visualized in Fig. 2, with alcohols and hydrocarbons dominating.
Within the alcohol group, 2-methyl-1-propanol, amylene hydrate, 2-butanol, 1-butanol and 3-methyl-3buten-1-ol were the new volatiles identified in A. titanum during this study. 2-Methyl-1-propanol (isobutanol), Table 1. Putatively identified volatile molecules that are statistically significantly different between the pre-flowering and the flowering phases. Data from 16 samples. Only peaks that were above the predefined detection criteria were included in the statistical calculations. a Statistically different volatiles in the pre-flowering versus the female flowering (PvF), the female versus the male (FvM), and the male versus the pre-flowering phases (MvP). The p-values were not corrected for multiple comparison adjustments. b Linear retention index was determined using C 8 -C 20 n-alkane standard solution on a Rxi-624Sil (60 m × 250 μm × 1.4 μm) column. RI below 800 was extrapolated by ChromaTOF. c Relative abundance = analyte total peak area / total peak area of all analytes detected. www.nature.com/scientificreports/ which smells like sweet alcohol 28 , was present at the highest relative abundance, followed by amylene hydrate (tert-amyl alcohol; camphoraceous odor 29 ), 3-methyl-3-buten-1-ol (isoprenol; fruity odor), 2-butanol (fruity odor) and 1-butanol (fruity odor). Various species of social wasps in the Vespula and Dolichovespula genera (yellow jackets) are attracted to 2-methyl-1-propanol, and the attraction extends to paper wasps (Polistes fuscatus) when 2-methyl-1-propanol is combined with acetic acid 30 . 3-Methyl-3-buten-1-ol (isoprenol) is a known metabolite of isopentenyl pyrophosphate, an isoprenoid precursor in the biosynthesis of terpenes and terpenoids in plants 31,32 . 2-Butanol and 1-butanol have been reported as the main constituents in the odor profile of A. pilosus and A. henryi, respectively 20,33 . 2-Butanol is a known aggregation pheromone of the male rhinoceros beetle, a coconut tree pest 34 . It has also been found as one of the attractants for two beetles, Parastasia bimaculata and Chaloenus schawalleri (Chrysomelidae) 35 . P. bimaculata is known to be attracted to heat-generating, odor-producing flowers, whereas C. schawalleri is less selective about the heat-generation. 1-Butanol is among the most abundant molecules in Petunia integrifolia and P. secreta pollens that attracts short-tongued bees of family Halictidae 36 . The hydrocarbons are the second largest group among the named molecules. The long-chain n-alkanes (C13, C14, C25 to C28) have been reported as being produced by the female flowers of A. titanum previously 37 . This is, however, the first time the volatile presence of the branched chain hydrocarbons (Table 1) is being reported for A. titanum, which includes 2-methylhexane, 2,4-dimethylhexane, 2,4-dimethyl-1-heptene, 3-methylhexane, 2,4-dimethylheptane and 2-methyl-1-pentene. Some of these branched hydrocarbons have, however, been detected as volatiles produced by other plants 38 Among ethers, ketones, esters and N-containing molecules, tetrahydro-2,2,5,5-tetramethylfuran, 2-methyl-2-(1-methylethoxy)propane, 2-methyl-1-propyl formate, 2-propenenitrile and 2-pentanone were the new molecules identified in A. titanum. Tetrahydro-2,2,5,5-tetramethylfuran is a known volatile molecule produced by the fungi Cladosporium cladosporioides that promotes plant growth 42 . 2-Propenenitrile has been detected as released by enophytic fungi, showing antifungal activity against Monilinia fructicola, a fungal plant pathogen 43 . It is possible that these two molecules monitored here were produced by microorganisms or perhaps produced by the plant itself. 2-Pentanone is the only ketone identified in the male flowering phase, and has been reported in the inflorescence odors of other Amorphophallus species, such as A. borneensis, A. eichleri and A. obscurus 44 .
The statistical analysis between the male flowering and the pre-flowering samples showed that 74 features were statistically significantly different (p ≤ 0.05). Of these, two features were overlapping with those found statistically significantly different between the pre-flowering and the female flowering samples, 57 features were overlapping with the statistically significantly different features between the female flowering and the male flowering samples, www.nature.com/scientificreports/ and 15 features were found new in this comparison. Of the 74 features, putative names could be assigned to seven molecules (Table 1) which were present at higher amounts in the male flowering phase, with alcohols and hydrocarbons dominating. Among these molecules, 2-butanol, 3-methylhexane, 2-methyl-2-(1-methylethoxy) propane, 3-methyl-3-buten-1-ol and 2,4-dimethylheptane were also found dominant in the male flowering phase during the comparison with the female flowering phase. 2-Methyl-1-pentene and 3-methyl-1-butanol were the additional molecules found dominant in the male flowering phase, and have been detected for the first time in the volatile profile of A. titanum. 2-Methyl-1-pentene was present at the highest relative abundance among all the hydrocarbons. 3-Methyl-1-butanol (isoamyl alcohol; fruity odor) has been reported previously in the inflorescence odors of A. borneensis, A. commutatus and A. konjac, as well as Aristolochia microstoma flowers 44,45 . Some studies have shown that the inflorescence of a plant produces different smells when injured 46 . In this study, before sample 8 was collected, a small window was cut into the bottom of the spathe of A. titanum for the greenhouse staff to conduct hand pollinations (Fig. 1e). After the window was made, production of the S-containing molecules and aldehydes dropped sharply, though they increased subsequently. The intensity of most of the alcohols and hydrocarbons increased instead, before being reduced to much lower levels, and rising again during the male flowering phase. It was not clear whether the injury was the cause for the sudden change of volatiles in sample 10.

Other volatile features of interest.
In addition, 304 other features were present throughout the flowering period of A. titanum, with hydrocarbons, aldehydes and ketones dominating. These statistically non-significant molecules, also likely contribute to the odors emitted during the pre-flowering and the flowering phases. Putative names could be assigned to 19 molecules in this group as shown in Table 2, of which 14 molecules were found for the first time in A. titanum during this study. These include, benzene, octane, 3-tridecene, butyl benzene, pentyl benzene, hexyl benzene, hexanal, octanal, pentanal, heptanal, acetone, 2,3-butanedione, 2-butanone and tetrahydrofuran.
The hydrocarbons mainly consisted of n-alkanes and alkylated benzenes (cyclic hydrocarbons), with toluene present at the highest abundance (Table 2). Toluene and heptane have been previously found in the scent of A. titanum inflorescence, with glue solvent-like and gasoline-like smell, respectively 9 . Toluene was also detected in the pollen aroma of Petunia integrifolia and Petunia secreta 36 . Benzene has been detected, in a low amount, in the floral scent of Gelsemium sempervirens 47 . Octane has been found among the aroma components of sweet cherry (Prunus avium L.) flower essential oils 48 . 3-Tridecene and pentylbenzene have been reported in the essential oil of Haplophyllum tuberculatum leaves and stem 49 . Butylbenzene has been detected in the volatile profiles of Gentiana asclepiadea flowers 39 . Pentylbenzene is reported as a component in the essential oil of aerial parts of Kellusia odoratissima (wild celery) ecotypes 50 .
Among the aldehydes, 2-propenal (acrolein) was present at the highest abundance (Table 2). It has been found as one of the indicators of A. titanum inflorescence blooming 51 . Hexanal (citrus, orange odor 52 ) has been reported as a minor volatile molecule in A. pilosus 33,44 . Octanal, along with heptane, 3-methylhexane, nonanal, octanal and other volatiles, has been identified as a semiochemical produced in Gossypium hirsutum for protection against Table 2. Putatively identified volatile molecules that are statistically non-significant between the pre-flowering and the flowering phases. The p-values were not corrected for multiple comparison adjustments. a Linear retention index was determined using C 8 -C 20 n-alkane standard solution. RI below 800 was extrapolated by ChromaTOF. b Relative abundance = analyte total peak area/total peak area of all analytes detected. www.nature.com/scientificreports/ predatory Chrysoperla species 41 . Octanal has also been found in the essential oil of H. tuberculatum aerial parts, along with 3-tridecene and pentylbenzene 49 . Pentanal (valeraldehyde) has been found in 'Fuego' variety of rose 40 , along with hexanal, heptanal and octanal among other volatile molecules. Butanal (butyraldehyde; rancid, sweaty odor) has been reported in the floral odors from A. titanum 23 . Heptanal (citrus, orange odor 52 ) is released in caterpillar-infested plant, Arabidopsis thaliana, along with 1-butanol, 2-pentanone, nonanal, heptanal and other volatiles, to attract Cotesia rubecula parasitoid in the defense mechanism against Pieris rapae caterpillars 53 . Acetone was found at the highest abundance among all the statistically non-significant molecules (  33,44 . 2-Butanone has been identified as a scent molecule in A. konjac 33 . 2,3-Butanedione (butter-like odor), along with heptanal and hexanal, has been found among the contributors to Symplocarpus renifolius floral scent during the female, bisexual, and male flowering phases 52 . Ethanol was the only alcohol identified throughout the flowering phase of A. titanum in the present study. It was previously found in the scent profile of other Amorphophallus species, such as A. cirrifer, A. konjac and A. obscurus 33,44 . Ethanol has also been detected among the major volatiles present in the pollen aroma of Petunia integrifolia and Petunia secreta 36 . Study limitations. Samples in this study were from only one flowering A. titanum which was located in a greenhouse filled with other plants, a small number of which were concurrently flowering. In this case, isolating the plant from others was not possible, however, once the plant started flowering, we are reasonably confident that the sampling was dominated by A. titanum sources based on the proximity of the sampler and the natural convection from thermogenesis. We note also that the greenhouse was illuminated by a warm, strong (100 W) light bulb each night, which may have impacted the plant's behavior. Importantly, while we reached level 2 of the naming convention outlined by the Metabolomics Standards Initiative (MSI) 54,55 , the molecules listed herein are only putative identities, which need to be verified using authentic standards.

Conclusion
The present study represents the first time series analysis of the volatile molecules emitted by A. titanum using GC × GC-TOFMS. By analyzing 16 samples collected over the flowering period, 422 volatile features were detected. Of these, 118 features were statistically significantly different between the pre-flowering phase and both flowering phases. A total of 26 of these molecules were assigned with putative identifications based on their mass spectral matching, of which 18 molecules are first time ever reported in A. titanum. The female flowering phase was dominated by the S-containing molecules and aldehydes, and the alcohols and hydrocarbons were dominant during the male flowering phase. While the S-containing molecules are the major contributors to the foul odor emitted during the female flowering phase, the aldehydes, alcohols, hydrocarbons and ketones also contribute to the unpleasant odor of the corpse flower. Some of these volatile molecules have been reported to play an important role in plant-insect interactions, and could be responsible for attracting the pollinators and inducing the defense mechanisms in A. titanum. The additional 304 features were found present during the entire flowering period, and 19 of these molecules were assigned putative names which also likely contribute to the putrid odor of the corpse flower. Of these 19 molecules, 14 molecules are the newly identified in A. titanum in this study. This study demonstrated that A. titanum not only emitted odorants in the female flowering phase, but also a large number and wide variety of volatile molecules in the male flowering phase.

Data availability
The datasets generated and/or analyzed during the current study are available on reasonable request to the corresponding author.